| 2007 |
PCFT (SLC46A1) is an electrogenic, proton-coupled folate transporter with optimal transport activity at low pH (pH 5.5), mediating intestinal folate absorption; transport is pH-dependent with increasing Km and decreasing Vmax as pH rises toward neutral, and transport in Xenopus oocytes is electrogenic and pH-dependent. |
Xenopus oocyte expression with electrophysiology, radiolabeled folate uptake assays, tissue fractionation showing apical brush-border membrane localization in duodenum/proximal jejunum |
American journal of physiology. Cell physiology |
High |
17340171 17898134 18174275
|
| 2007 |
Loss-of-function mutations in PCFT (SLC46A1) are the molecular basis for hereditary folate malabsorption (HFM); mutations cause decreased protein stability, defects in membrane trafficking, or loss of transport function, with some mutants retaining residual activity. |
Sequencing of PCFT gene in HFM patients, transient transfection of mutant constructs in HeLa cells with transport assays, Western blot for protein stability, membrane trafficking assessment |
Blood |
High |
17446347
|
| 2008 |
PCFT co-localizes with folate receptor alpha (FRα) in the endosomal compartment and plays a functional role in FRα-mediated endocytosis by serving as the route of export of folates from acidified endosomes into the cytosol; probenecid inhibits PCFT-mediated transport at endosomal pH and blocks FRα-mediated folate delivery to the cytosol. |
FRα cDNA transfection into PCFT(+) and PCFT(−) HeLa sublines, radiolabeled folate transport assays, immunofluorescence co-localization, probenecid inhibition experiments |
The Journal of biological chemistry |
High |
19074442
|
| 2008 |
PCFT (SLC46A1) is N-glycosylated at two canonical asparagine residues; the protein migrates at ~55 kDa on SDS-PAGE (predicted ~50 kDa) due to glycosylation, collapses to ~35 kDa after deglycosylation. Single glycosylation-site mutants retain full transport activity; the double deglycosylation mutant retains a majority of activity. The N- and C-termini are intracellular (accessible only in permeabilized cells). |
PNGase F treatment, tunicamycin treatment, site-directed mutagenesis of glycosylation sites (Asn→Gln), Western blot, HA-tag accessibility assay in permeabilized vs. intact cells |
Biochimica et biophysica acta |
High |
18405659
|
| 2009 |
His247 and His281 residues are functionally critical for PCFT (SLC46A1). His247 (located in the large loop between TM6 and TM7 at the cytoplasmic entrance, in hydrogen-bond distance to Ser172) is required for substrate selectivity and coupled proton transport; H247A mutation increases proton 'slippage' (folate-independent proton transport) and reduces substrate specificity. His281 (in TM7, extracellular face) facilitates proton binding/protonation that augments folate binding; H281A increases folic acid influx Kt ~12-fold but does not abolish proton coupling. |
Site-directed mutagenesis, radiolabeled folate influx kinetics in transfected HeLa cells, electrophysiology in Xenopus oocytes, homology modeling |
The Journal of biological chemistry |
High |
19389703
|
| 2009 |
PCFT promoter silencing through CpG hypermethylation within the minimal transcriptional regulatory region (−42 to +96 bp from TSS) accounts for loss of PCFT expression in antifolate-resistant HeLa R1-11 cells; treatment with 5-aza-2'-deoxycytidine restores transport and PCFT mRNA. In vitro methylation of the transfected reporter plasmid inhibits expression. Gene copy loss contributes additively. |
Bisulfite conversion sequencing, luciferase reporter assays, 5-aza-2'-deoxycytidine treatment, in vitro methylation of reporter plasmid, FISH for gene copy number |
Molecular cancer therapeutics |
High |
19671745
|
| 2009 |
Glu185 (E185) in a PCFT transmembrane domain plays a critical role in proton-folate coupling: E185A-PCFT loses function at pH 5.5 (8-fold decrease in Vmax) but retains function at pH 7.4; the transport remains intrinsically functional (capable of trans-stimulation). The effect of E185 substitution is primarily on the maximal transport rate (Vmax), consistent with involvement at a rate-limiting step of the PCFT transport cycle. |
Site-directed mutagenesis, radiolabeled methotrexate influx kinetics, trans-stimulation assays, surface biotinylation in transfected HeLa cells |
American journal of physiology. Cell physiology |
High |
19403800
|
| 2010 |
PCFT secondary structure consists of 12 transmembrane domains with both N- and C-termini directed to the cytoplasm, established by substituted cysteine accessibility method (SCAM). A disulfide bridge between native Cys66 and Cys298 (in the 1st and 4th extracellular loops) is present but not required for transport function. |
Substituted cysteine accessibility method (SCAM) with MTSEA-biotin, streptavidin pulldown, Western blot, beta-mercaptoethanol treatment, cysteine-less PCFT generation |
Biochemistry |
High |
20225891
|
| 2010 |
Asp156 (D156) is critical for PCFT protein stability; multiple substitutions at this site (Tyr, Trp, Phe, Val, Asn, Lys) result in protein instability and loss of function, while conservative substitutions (Glu, Gly) preserve substantial function correlating with surface expression. Asp109 (D109), located in the first intracellular loop between TM2 and TM3, is absolutely required for PCFT transport function regardless of pH or substrate concentration, despite normal surface expression. |
Site-directed mutagenesis, radiolabeled folate transport assays at multiple pH values and concentrations, surface biotinylation, Western blot for protein stability in transfected HeLa cells |
Blood |
High |
20805364
|
| 2010 |
NRF-1 (nuclear respiratory factor 1) is a major transcriptional regulator of PCFT (SLC46A1). NRF-1 binds to three consensus sites in the PCFT minimal promoter; NRF-1 overexpression increases PCFT mRNA and reporter activity, while dominant-negative NRF-1 or NRF-1 siRNA markedly represses PCFT expression. |
EMSA with NRF-1 antibody supershift, chromatin immunoprecipitation, luciferase reporter assays with NRF-1 site mutations, NRF-1 overexpression and siRNA knockdown |
The Journal of biological chemistry |
High |
20724482
|
| 2010 |
Arg376 (R376) is important for proton binding in PCFT; R376Q and other non-positive substitutions impair proton binding, which in turn modulates the folate binding pocket and reduces the rate of carrier conformational change. The R376Q mutant retains folate-independent proton transport (slippage/channel-like property) in Xenopus oocytes, but substrate transport is markedly decreased, with substrate-dependent differences. |
Site-directed mutagenesis, radiolabeled folate influx kinetics, electrophysiology in Xenopus oocytes, transfection in HeLa R1-11 cells |
American journal of physiology. Cell physiology |
High |
20686069
|
| 2011 |
PCFT-deficient (PCFT−/−) mice develop severe systemic folate deficiency confirming the critical and nonredundant in vivo role of PCFT in intestinal folate transport; PCFT deficiency causes macrocytic normochromic anemia, pancytopenia, impaired erythroblast differentiation with increased apoptosis, elevated erythropoietin, soluble transferrin receptor, and thrombopoietin. |
Targeted gene disruption (knockout of exons 1–3), in vivo folate uptake experiments, hematological analysis, bone marrow histology, serum cytokine measurement |
Blood |
High |
21346251
|
| 2011 |
PCFT and RFC (reduced folate carrier) are distributed in lipid rafts of the colonic apical membrane; chronic ethanol ingestion reduces PCFT and RFC protein levels in lipid raft fractions of the colon, associated with decreased folate transport affinity and Vmax. |
Optiprep density gradient floatation for lipid raft isolation, Western blot, radiolabeled folate transport in apical membrane vesicles from ethanol-fed rats |
Journal of cellular physiology |
Medium |
21069807
|
| 2012 |
Gly189 and Gly192 (a GxxG motif in TM5) are functionally critical for PCFT; alanine substitution abolishes function. Ile188, adjacent to this motif, is accessible to the PCFT aqueous translocation pathway and located in the folate binding pocket (substrate protection of I188C from sulfhydryl-reactive reagents is retained at 0°C). Met193C is also aqueous-accessible but substrate protection is lost at 0°C. |
Site-directed mutagenesis, substituted cysteine accessibility method with membrane-impermeable reagents, substrate protection assays at different temperatures, radiolabeled folate transport |
American journal of physiology. Cell physiology |
High |
22785121
|
| 2012 |
Ala335 residue contributes to PCFT protein stability; substitutions rendering bulky or charged residues at this site cause protein instability and loss of function. Gly338 residue is required for PCFT conformational oscillation (cycling between transport states); G338C mutant retains substrate binding (minimal change in Ki) but has 15–20-fold decreases in Kt and Vmax and is inaccessible to MTS reagents, consistent with TM9 location and impaired carrier cycling rather than substrate binding defect. |
Site-directed mutagenesis, radiolabeled pemetrexed and methotrexate influx kinetics, surface biotinylation, SCAM with MTS reagents, homology modeling |
American journal of physiology. Cell physiology |
High |
22843796
|
| 2013 |
Bicarbonate at physiological concentrations produces potent and rapidly reversible inhibition of PCFT-mediated transport at neutral pH. Bisulfite and nitrite also inhibit PCFT, particularly at weakly acidic pH by decreasing Vmax and collapsing the transmembrane proton gradient. Sulfate, nitrate, and phosphate do not inhibit PCFT. |
Radiolabeled folate transport assays in PCFT-stable HeLa transfectants at varying anion concentrations and pH, intracellular pH measurements |
Molecular pharmacology |
Medium |
23609145
|
| 2013 |
KLF4 is the primary transcriptional activator of PCFT in the small intestine; HNF4α synergistically enhances KLF4-induced PCFT transcription. CDX2 and C/EBPα suppress KLF4- and KLF4/HNF4α-induced PCFT promoter activity. The graded expression pattern of these factors along the intestinal tract accounts for the proximal-restricted expression of PCFT. |
Dual-luciferase reporter assays in HEK293 cells with cotransfection of transcription factors, Western blot for transcription factor expression along rat intestine |
Biochemical and biophysical research communications |
Medium |
23313509
|
| 2015 |
Four Tyr residues (Y291, Y362, Y315, Y414) are extracellularly accessible in PCFT; Y291, Y362, and Y315 are located within or near the folate binding pocket. Substitution of these residues with Cys or Ala increases both influx Vmax and Kt/Ki (suggesting release from a high-affinity constrained state), while Phe substitution moderates these changes. Y315A PCFT shows increased Vmax but loss of transstimulation (exchange), indicating these Tyr residues constrain carrier mobility and secure the high-affinity substrate-binding state at the expense of oscillation rate. |
SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled influx and efflux transport kinetics, transstimulation assays |
American journal of physiology. Cell physiology |
High |
25608532
|
| 2016 |
Trp299 (W299) in the 4th external loop between TM7 and TM8 is required for PCFT function through its hydrophobicity: W299S mutation decreases Vmax 6.5-fold and alters Kt/Ki; only Phe and (partially) Ala substitutions preserve function, suggesting this residue interacts with the lipid membrane during the transport cycle and influences carrier oscillation and the folate binding pocket indirectly. |
SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled pemetrexed transport kinetics, DTT treatment for disulfide accessibility |
American journal of physiology. Cell physiology |
Medium |
27251438
|
| 2017 |
Purified human PCFT expressed in Sf9 insect cells via baculovirus retains folate transport function after reconstitution into liposomes, enabling biochemical and structural studies. |
Baculovirus/Sf9 expression, detergent purification, reconstitution into liposomes, radiolabeled folic acid transport assay |
PloS one |
Medium |
28493963
|
| 2017 |
The 8th transmembrane helix of PCFT defines the aqueous translocation pathway: 14 contiguous exofacial residues are accessible to MTSEA-biotin; pemetrexed blocks biotinylation of 6 deep residues implicating this region in folate binding. Pro314 and Tyr315 are critical for function; their substitution markedly increases Kt, Ki, and Vmax. Homology modeling (based on GLUT5 structures) predicts a helix break in the outward-open conformation at this region. |
SCAM with MTSEA-biotin, site-directed mutagenesis, substrate protection assay, temperature-dependence of accessibility, homology modeling based on GLUT5 structures |
Biochimica et biophysica acta. Biomembranes |
High |
28802835
|
| 2017 |
The 7th and 8th transmembrane helices of PCFT form an exofacial cleft defining the aqueous translocation pathway; 9 exofacial residues of TM7 are accessible. Paired cysteine substitutions between TM7 and TM8 (T289C-I304C and Q285C-Q311C) spontaneously form disulfide bonds, impairing accessibility and function; DTT restores both, establishing close proximity of these helices and their role in the substrate entry pathway. |
SCAM, dicysteine cross-linking with spontaneous disulfide bond formation, DTT reversal, homology modeling based on GLUT5 inward- and outward-open conformations |
American journal of physiology. Cell physiology |
High |
29167151
|
| 2018 |
Asn411 (N411) in TM11 is part of the external gate of PCFT; N411K mutation causes loss of function with a markedly decreased influx Vmax and reduced affinity for most folate substrates (especially 5-methyltetrahydrofolate). Positive-charged substitutions are most disruptive; negative (Asp) and bulky hydrophobic/polar substitutions are better tolerated. Homology modeling places N411 protruding into the aqueous pathway, most prominently in the inward-open conformation. |
Site-directed mutagenesis, surface biotinylation, radiolabeled folate transport kinetics, homology modeling based on GLUT5 |
Blood advances |
High |
29344585
|
| 2018 |
PCFT generates substantial transmembrane electrochemical-potential gradients (concentrative transport) for antifolates at extracellular pH levels relevant to the tumor microenvironment (pH 6.9–7.0). Concentrative transport requires the transmembrane proton gradient; it is abolished by the protonophore CCCP and is not observed in bicarbonate/CO2-buffered medium at neutral pH. |
Radiolabeled methotrexate accumulation assays in PCFT-expressing HeLa cells, CCCP treatment, Na+/H+ exchanger inhibitors, intracellular pH measurement |
Molecular pharmacology |
Medium |
29326243
|
| 2019 |
Asp109 (D109) in the first intracellular loop locks PCFT in an inward-open conformation when substituted (D109A); cysteine-substituted residues in the aqueous translocation pathway lose accessibility to MTSEA-biotin. Introduction of a second 'unlocking' substitution at Gly305 (G305L or bulky residues in TM8) in the D109A scaffold largely restores both function and aqueous accessibility, demonstrating that D109 is part of a 'motif A' (GXXXDXXGR) critical for carrier oscillation between conformational states. |
Site-directed mutagenesis, SCAM with MTSEA-biotin, functional transport assays, double-mutant epistasis analysis, homology modeling |
The Journal of biological chemistry |
High |
30858177
|
| 2020 |
SLC46A1 regulates hepatic iron metabolism by importing heme into hepatocytes; hepatocyte-specific inhibition of SLC46A1 by AAV decreases liver iron content, increases serum iron and free heme, and alters expression of iron-regulatory molecules (TfR1, hepcidin, ferroportin). In hepatocytes, SLC46A1 can import hemin, increasing intracellular iron content; hemin import is independent of folate transport, but hemin treatment decreases SLC46A1 expression and folate import. |
AAV-mediated hepatocyte-specific SLC46A1 knockdown in mice, hemin uptake assay in hepatocytes, measurement of intracellular iron content, Western blot for iron-regulatory proteins |
Metabolism: clinical and experimental |
Medium |
32621820
|
| 2025 |
Molecular dynamics simulations based on cryo-EM structure of Gallus gallus PCFT (ortholog of hPCFT) reveal that HFM-causing mutations cause partial loss of structural integrity manifested as enlarged/distorted pore, loss of long-range contacts, less stable inner helices with reduced solvent accessibility, and loss of secondary structure; these structural changes are reversed by compensatory mutations that restore function. |
Molecular dynamics simulations of hPCFT homology model based on cryo-EM structure of Gallus gallus PCFT, structural analysis of disease-causing and compensatory mutations |
The Journal of biological chemistry |
Medium |
39924111
|
| 2008 |
PCFT/HCP1 transports both folate and heme, but with lower affinity for heme than folate; siRNA knockdown of PCFT/HCP1 in Caco-2 cells reduces both folate uptake (48%) and heme uptake (22.5%), with greater impact on folate. Folic acid inhibits heme transport in hypoxic but not normal mice in vivo. |
siRNA knockdown in Caco-2 cells, radiolabeled folate and 59Fe-heme uptake assays, in vivo mouse intestinal transport studies with blocking antibody, everted duodenum preparations |
The British journal of nutrition |
Medium |
18782461
|
| 2022 |
Loss of SLC46A1 expression in hepatocellular carcinoma (HCC) cells causes iron deficiency in tumor tissue; only SLC46A1 (not STEAP3 or DMT1) silencing or overexpression controlled intracellular iron content in HCC cells. Lentivirus-mediated re-expression of SLC46A1 in orthotopic tumors restores tumor iron content with corresponding changes in iron-metabolic molecules. |
Lentiviral SLC46A1 overexpression and siRNA knockdown in HCC cell lines, orthotopic tumor implantation in mice, intracellular iron content measurement, Western blot for iron-metabolic molecules |
Hepatology communications |
Medium |
35811443
|
| 2026 |
SLC46A1-mediated folate uptake in colorectal cancer cells suppresses tumor proliferation, migration, and invasion; mechanistically, SLC46A1 deficiency reduces intracellular folate availability, impairs cellular methylation potential (decreased SAM/SAH ratio), causes DNA hypomethylation at the FOS proto-oncogene promoter, and transcriptionally activates CCND1, BCL2, and PLAU oncogenic effectors driving CRC progression. |
SLC46A1 loss/gain-of-function in vitro and in vivo, SAM/SAH ratio measurement, bisulfite sequencing/methylation analysis, ChIP for promoter methylation, transcriptomic analysis, tumor growth assays |
Cell death & disease |
Medium |
41620398
|